Codon-dependent translation connects late chemical evolution to biological evolution, which is the basis for phylogenetic inference and genetics. Aminoacyl-tRNA syntheses (aaRS) actually translate the code. Experimental study of ancestral aaRS is thus deeply and broadly valuable to contemporary molecular biology, genetics, biophysics, and biochemistry. Previous work created and extensively validated a new approach to accessing events and processes associated with the origins of translation. The experimental approach uses Urzymes, which are constructs developed from invariant cores of protein superfamilies'. Urzymes representing ancestral forms of Class I and II (aaRS) have ~15% of the mass but retain 60% of the catalytic proficiency of contemporary aaRS. The approach also introduced new bioinformatics to show that Class I and II aaRS descended from opposite strands of the same gene. Thus, codon middle-base pairing of multiple sense/antisense alignments (<MBP>) probes earlier phylogenetic relationships than those accessible using ancestral gene reconstruction. This proposal will exploit these new tools to address three questions: (I) What molecules and processes gave rise to the highly evolved sophistication of Class I and II Urzymes? <MBP> values for 94-residue Class I and II aaRS Urgenes will distinguish the order in which the two strands specialized, breaking the constraint of sense/antisense coding, and radiated to give subclasses and then a full canonical set of coded amino acids. Catalysis by 46-aa ATP binding sites from a designed sense/antisense gene have been validated by mutagenesis. We will use thermodynamic cycles to measure coupling between 46mer active-site residues and attempt to construct them using reduced amino acid alphabets. (II) How do Urzyme structures and functions differ from contemporary enzymes? tRNA specificities and long-range energetic coupling will be measured in Urzymes and in Class II HisRS. NMR studies will determine whether the Uryzmes are folded, molten globular or intrinsically disordered; the temperature dependence of stability and catalysis will verify the resulting conclusions. (III) What biological roles could the Urzymes have carried out in the course of implementing the genetic code? A novel method will use construction of aaRS knockouts to test whether Urzymes can complement aaRS knockouts or whether they are toxic in vivo and an in vitro translation system will be developed, to eventually recapitulate steps in the development of the canonical genetic code from defined components. Previous results imply these are all doable.